Molecular oncology 2017 11 28() doi 10.1002/1878-0261.12160Abstract
The mitogen-activated protein kinase (MAPK) pathway plays a central role in colorectal cancers (CRC). In particular, BRAF V600E-mutant tumors, which represent around 10% of CRCs, are refractory to current therapies. Over-expression and secretion of serine peptidase inhibitor Kazal type 1 (SPINK1) is observed in around 50% of CRCs and its serum level can be used as a biomarker for poor prognosis. Utilizing a recently developed Extendable Blocking Probe assay, we analyzed the BRAF mutation status in a CRC patient cohort (N=571) using tissue-derived RNA as the starting material. From the same RNA samples we measured the relative SPINK1 expression levels using a quantitative real-time PCR method. Expression of mutant BRAF V600E correlated with poor prognosis, as did low expression of SPINK1 mRNA. Further, BRAF V600E correlated negatively with SPINK1 levels. In order to investigate the effect of MAPK pathway targeted therapies on SPINK1 secretion, we conducted in vitro studies using both wild-type and V600E colorectal cancer cell lines. BRAF inhibitor vemurafenib, and subsequent MAPK pathway inhibitors trametinib and SCH772984,significantly increased SPINK1 secretion in V600E CRC cell lines Colo205 and HT-29 with concomitant decrease in trypsin-1 and -2 secretion. Notably no SPINK1 increase or trypsin-1 decrease was observed in BRAF wild-type CRC cell line Caco-2 in response to MAPK pathway inhibitors. In further mechanistic studies, we observed that only trametinib was able to diminish completely both MEK and ERK phosphorylation in the V600E CRC cells. Furthermore, the key regulator of integrated stress response, activating transcription factor 4 (ATF-4), was downregulated both at mRNA and protein level in response to trametinib treatment. In conclusion, these data suggest that sustained inhibition of not only MAPK pathway activation, but also ATF-4 and trypsin, might be beneficial in therapy of BRAF V600E-mutant CRC and that SPINK1 levels may serve as an indicator of therapy response.